It is the usual practice to employ trained medical personnel to obtain samples and cultures for analysis from body cavities, such as the vagina, rectum, nose, sinuses, ears, mouth or throat. Typically sample collection is performed using a swab, brush, or the like.
Care must be taken in collecting a sample from the area of interest in order to avoid contamination of 1) the sample; 2) the collector of the sample; or 3) the donor of the sample. Care must also be taken to minimize the chance of injury to delicate cavity areas. However, even if trained medical personnel perform the sample collection, broad variability in sample quality and yield and in reproducibility is observed when currently available sample collection devices are employed. Furthermore, sample collection devices currently in use are not typically adaptable for use in collecting samples from small subjects, such as neonatal laboratory animals.
Obtaining cells from neonatal rodents is critical for investigations involving genetically-modified (e.g., transgenic and knockout) mice and rats. Various practices exist for obtaining cells from animals for DNA genotyping. For example, common practices used to acquire cells from a neonatal mouse for DNA genotyping involve total or partial amputation of the rodent's toe, ear or tail. These practices are both invasive and mutilating, and toe clipping has been generally banned.
In response to these practices, mice exhibit responses to pain that may include one or more specific behaviours such as vocalizing, biting and avoidance (running away). Neonatal mice contort and move their entire bodies back and forth in response to tail clipping, showing that they experience significant pain. Following amputation of tail tissue, some mice bleed to death or succumb to cannibalization.
Non-invasive and painless buccal cell isolation methods involving saliva, mouthwash, treated filter paper, cytobrush and foam or cotton-tip swabs are available for collecting buccal cells from humans for diagnostic analyses. These methods, however, are extremely difficult, if not impossible, to adapt for the small size of, for example, the neonatal and juvenile mouse. For example, the filter paper on the Bode Buccal DNA collector is about the width of a neonatal mouse head. The Oragene®•DNA Self Collection Kit (DNA Genotek Inc.) requires 1-2 ml of human saliva sample to extract sufficient DNA for processing, or approximately the total weight of the 1 g neonatal mouse. Traditional cotton swabs are much too large to collect buccal cells from the neonatal mouse and would have the potential to suffocate the mouse if forced into the mouth. Although comparison of cytobrush, mouthwash and treated card for obtaining human buccal cells found that the cytobrush was the best method for human sampling, these approaches cannot be adapted directly to mice, especially day of life 1-15 mice. A serrated pipette tip used to collect human cells for RNA isolation is too abrasive for the fragile neonatal mouse cheek. Mouse pups require methods of handling that minimize pain and stress, since they are very fragile and can die easily during execution of the experimental protocol, succumb to cannibalization by the dam following the sampling, or succumb to starvation following rejection by the dam.
Protocols have been developed to address minimally invasive and painless mouse buccal cell sampling. One method for sampling and extraction of mouse DNA used a cotton swab adapted for adult, but not neonatal, mice. A common toothpick, though more appropriate in size, would lack a reservoir for collecting adequate buccal cells from the newborn mouse for subsequent analyses. Kits involving a buccal brush or swab (Epicentre), mouse saliva (10 μL; Sigma), and buccal cells applied on a card with a swab (Whatman) are examples of commercial products available for adult mice. However, the youngest mouse described in the protocols for these products was 1 month old. Due to alterations in their genotype, it is not uncommon for knockout and transgenic mice to die within this time frame. The present invention and method allows for early sampling of genetic material from neonatal mice, enabling researchers to identify and possibly treat transgenic and knockout mice within their first few days of life.
Recently, a scoop-shaped tool has been developed for sample collection from the mouth of a neonatal mouse (Zhang et al., WO 2007/109586 A2 and US 2009/0075289). While the scoop-shaped tool addresses some of the problems of the past, sample collection using this scoop requires technical skill in order to obtain reproducibly good quality samples.
Other sample collection devices are described in US2007/0249961 and US2003/0181826, for example.
To date, known, standardized, relatively non-invasive methods for buccal cell sampling from neonatal mice are not available from commercial sources or disclosed in published literature.
Therefore, there remains a need for a non-invasive, non-mutilating approach to obtain cells and biomolecules from animals, such as neonatal mice, for experimental procedures that require an oral sample, such as buccal cell sampling, for example for DNA extraction and genotyping, RNA expression analysis, detection of disease or detection of an infectious agent (e.g., virus, bacteria, fungus).
Furthermore, there remains a need for a sample collection tool that requires only minimal technical skill to obtain reproducibly good sample quality from body cavities, such as the vagina, rectum, nose, sinus, ears, mouth or throat. Good sample quality is generally determined by a sufficient, reproducible yield of biomolecules, such as nucleic acids and proteins, to readily permit downstream analysis. Such a tool would ideally also be amenable for use with standard collection and analysis containers and tools, such as sample collection receptacles, assay tubes and pipettes.